Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading

Biotechnological plastic recycling has emerged as a suitable option for addressing the pollution crisis. A major breakthrough in the biodegradation of poly(ethylene terephthalate) (PET) is achieved by using a LCC variant, which permits 90% conversion at an industrial level. Despite the achievements, its applications have been hampered by the remaining 10% of nonbiodegradable PET. Herein, we address current challenges by employing a computational strategy to engineer a hydrolase from the bacterium HR29. The redesigned variant, TurboPETase, outperforms other well-known PET hydrolases. Nearly complete depolymerization is accomplished in 8 h at a solids loading of 200 g kg−1. Kinetic and structural analysis suggest that the improved performance may be attributed to a more flexible PET-binding groove that facilitates the targeting of more specific attack sites. Collectively, our results constitute a significant advance in understanding and engineering of industrially applicable polyester hydrolases, and provide guidance for further efforts on other polymer types.

this method should be stated.In contrast, the GRAPE strategy, despite its limited novelty, has suggested four of the eight useful residue substitutions in TurboPETase, which appears to be more effective for universal use.In this context, as described in lines 267 and 268, the A251C/A281C double mutations should form a disulfide bridge that is distinct from those introduced to LCC-ICCG and many other PETases at an alternative position.I am unable to find experimental evidence that the A251C/A281C disulfide bridge is actually formed.This must be demonstrated using structural biology techniques or biochemical methods.3.) In Figure 2, the authors could present new experimental data for comparing different PETases under optimal buffer and temperature conditions.In general, lower ionic strengths and buffer concentrations are used in the new experiments, which are also related to the final depolymerization experiments with high substrate solid loading.Nonetheless, based on the information provided in the supplementary information, 1.7(1) and 1.10, the comparison of the activity of various mutants (Fig. 1D) and kinetic analysis (Figure 3 and Table 1) still only included data collected with 1 M potassium buffer, which can significantly (rather positively) influence enzyme activity, stability, and adsorption behavior to the substrate (, and this is widely accepted by scientific communities).This may render the mutant ranking invalid and result in incomparable kinetic properties to the degradation experiments depicted in Figures 2 and 4. The ideal solution is to repeat the kinetic analysis at lower buffer concentrations and, at the very least, probe several mutants shown in Fig. 1D to ensure that the use of 1 M buffer has no (negative) effect on their ranking in enzyme activity comparison.Furthermore, neither the main text nor the supplementary information explains how and in which buffer the Tm of individual mutants was determined.As previously stated, if 1 M buffer was used, certain mutants could be sufficiently thermoactivated and stabilized.Therefore, the superior mutant TurboPETase deduced based on the data in Fig. 1D and the supplementary tables may not be the ideal one to be used in a buffer-free condition (Fig. 4B).Comparing the old datasets (which have been moved to Fig. S4) with the new datasets in Fig. 2, it was also observed that the use of 1 M buffer can improve the degradation performance of TurboPETase by approximately 4-fold (at 50°C) or 7-fold (at 65°C) at similar enzyme to substate ratios but low solid loading levels.From an opposing perspective, have the authors considered confirming this benefit of using a high buffer concentration for large-scale, high-solid-loading experiments such as those depicted in Figure 4? For a future industrial application in the real world, a high buffer concentration will undoubtedly raise numerous (such as cost-related) questions.However, it would be of great scientific interest to determine if a 1 M buffer will further increase the degradation efficiency of TurboPETase and, if so, to discuss whether it would be preferable to use a low salt concentration at the expense of drastically reducing the degradation efficiency.4.) The depolymerization performance with TurboPETase, as shown in Figure 4, and the maximum production rate described in line 289, are unquestionably the highlights of this study.However, based on the polymer property data available thus far, the PET substrate used in this study has a crystallinity of 10.9%, which is significantly lower than the 14.6% used by Tournier et al (2020) with LCC ICCG.The final crystallinity of residual PET materials at 65°C as a result of physical aging and degradationinduced crystallization is in the range of 12.3-12.8%,which is still significantly lower than the starting material used by Tournier et al.As a result, I believe that the remarkable degradation performance should rather be attributed to the more "heavily amorphized" waste PET in comparison to the less amorphized materials used by Tournier et al. Furthermore,ref. 32.indicated that the molecular weights of the pretreated PET waste may influence their degradability; thus, the authors should provide GPC analysis of their pristine PET waste, those after extrusion, after micronization, and after enzymatic degradation individually for a better understanding of the correlation between polymer property and degradability.5.) The inferior performance of LCC ICCG at 65°C shown in Fig. 4 appears plausible to me, consistent with the data shown in Fig. 2 and also by Tournier et al., who stated that LCC ICCG should perform similarly to wt LCC at 65°C.However, the marginally better-performed degradation curve with LCC ICCG at 72°C with a similar shape to that determined at 65°C until at least 14 h did not appear to be reasonable.This is in stark contrast to previous studies such as those by Tournier et al.,Zeng et al. (ref. no. 30), and this recently published one by Ding et al. (https://doi.org/10.1016/j.jhazmat.2023.131386)also focusing on further protein engineering of LCC ICCG.All previous studies clearly demonstrated a significantly higher (~1.5 to 2-fold) degradation activity of LCC ICCG at temperatures ranging from 72-74°C compared to those at 65°C to 66°C.Given the lower crystallinity of the substrate used in this study (10.9%), the degradation performance with LCC ICCG at 72°C did not appear to be reliable or reproducible (i.e. should be much higher).Furthermore, as shown in Fig. S1B, the crystallinity of the PET substrate at 72°C exceeded 20% after 4 hours of reaction; however, it did not appear that the LCC-ICCG reaction rates were significantly reduced before 12 hours.The degradation curve at 72°C behaved in some ways like a "parallel" curve with the one determined at 65°C, where the crystallinity of the PET substrate continuously maintained less than 15% in correlation to a high degradability.This makes absolutely no sense to me.6.)I observed that the degradation curves in Fig. 4A and Fig. S6 were calculated using HPLC analysis, whereas the one depicted in Fig. 4B was determined using only NaOH consumption.I believe that these data ought to be better validated, similar to those presented by Tournier et al. in their extended Fig. 5 to calculate normalized depolymerization curves based on combinatorial data obtained by NaOH consumption, HPLC analysis of the formation of TPA/MHET/BHET; EG, and the weight loss determinations, with the original data presented separately in a table.7.) I am unable to fully comprehend the statement between lines 299 and 301, which I find to be quite confusing.I believe it is necessary for the authors to provide data and more thorough descriptions of the "water constraint" and "supersaturated TPA" concentration profiles under various reaction conditions.8.) Statements in lines 20-21 and 356-357 appear to be exaggerated.At least in refs.30 and 32, as well as the omitted publication by Ding et al. (see above for DOI), additional enzymes or mutants that outperform LCC-ICCG under specific reaction conditions, have been demonstrated.Obviously, these enzymes are not included in this study for experimental comparison with TurboPETase, nor are they even given serious consideration.As a highly active research field, additional promising enzyme variants may be published during this manuscript's revision stage.Before overselling their own findings, I strongly advise the authors to remain open to learning about the most recent developments in this research field.
Reviewer #3 (Remarks to the Author): The manuscript entitled Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading provides some interesting information related to enzyme development.However, given the high number of articles published in this and other journals related to incremental improvements of PET hydrolyzing activity of various enzymes, the novelty of this article seems to be very limited.The authors tried to compare the activity of their enzyme to some recently published PET hydrolyzing enzymes while it is not clear how e.g.different reaction conditions or enzyme thermostabilities may affect the comparison in figure 4. Apart from decomposition of PET, a lot of other parameters would be interesting to be included in a more mechanistic comparison of different PET hydrolyzing enzymes, such as sorption characteristics, activity on small water soluble (model) substrates, release of oligomers and activity thereon, specificity for other aromatic / aliphatic polyesters, potential synergies with other enzymes etc.

General responses to the reviewers' comments
We sincerely appreciate the careful way the reviewers examined the manuscript and are pleased with the positive comments.Meanwhile, their constructive criticisms and valuable suggestions are of great help in refining the manuscript.Accordingly, extensive additional experiments were performed and the manuscript has been substantially revised.Here's a brief overview of the key revisions: (1) Adsorption experiments: We have added the adsorption experiments utilizing a simple Langmuir approach, offering deeper insights into the binding efficiency of TurboPETase to heterogeneous PET films.Steady-state kinetics on soluble substrates (MHET and pNPB) were also performed to allow for a more comprehensive discussion on the potential reasons for enhanced depolymerization capabilities of TurboPETase.
(2) Structural analysis: In the revised manuscript, we have expanded our analysis to the potential local structural changes induced by the mutations.The new insights may offer a more nuanced molecular understanding of TurboPETase's catalytic attributes.
(3) Hydrolysis efficiency under the request conditions: We introduced new kinetic analyses and tested a suite of 12 mutants based on the M6 scaffold under conditions using a 100 mM KH2PO4-NaOH buffer.Additionally, we evaluated the depolymerization of TurboPETase and LCC ICCG on high-crystallinity PET materials to address the concern raised by reviewer #2 about whether TurboPETase is only efficient on extremely low-crystallinity PET substrates.
(4) Extended applications: To explore the potential broader applications of TurboPETase, we assessed its synergistic effects with BHETase.Additionally, we conducted supplementary experiments on the depolymerization of another aromatic polyester, PBT.
(5) Comparative analysis: During our submission process, a series of works emerged on the engineering of PET-degrading enzymes.We selected the most recent and influential enzymes for further comparative analysis, with TurboPETase still outpacing.Nonetheless, we agree with the reviewer's comments and have revised our manuscript to TurboPETase is more superior compared to the well-known enzymes.
We deeply appreciate the reviewers' feedback, which prompted us to undertake supplementary experiments that substantially enhance the robustness and reliability of our study.The precise amendments are elaborated upon below and are highlighted in yellow in the revised manuscript.

Point-to-point responses to the reviewers' comments:
Reviewer 1: Comment 1: 1.Even though turboPETase outperforms previous PETase mutants, the increase in performance is not very substantial.I was not convinced that turboPETase is a breakthrough for plastic recycling.It represents a step forward on a mainly incremental work of enzyme optimization and not a novel solution or a radically improved solution.In this sense, I consider that the work does not have the level of novelty and relevance necessary for publishing in Nat.Methods.

Response:
We genuinely appreciate the reviewer's acknowledgment of the merits in our work.Over the last 3 years, we have witnessed a number of success in the engineering of PET hydrolases, gearing towards potential applications in industrial plastic recycling processes.However, a majority of works aiming at enhancing enzyme performance are performed in small-scale reactions, often at low enzyme and substrate concentrations, even though large-scale reaction experiments up to 200 g kg -1 substrate loading have been reported (Nature 2020, 580, 216).In heterogeneous catalysis reactions, different enzymes may exhibit dramatically distinct catalytic efficiencies at various enzyme and substrate concentrations, as in the case of cellulases (ACS Catal. 2017, 7, 4904-4914;Biotechnol Biofuels. 2020, 13, 58).For instance, Erickson et al. demonstrated that IsPETase showed similar hydrolytic conversion compared to its variant IsPETase W159H/S238F at low enzyme loading (0.5-1 mgenzyme gPET -1 ), but approximately 2-fold lower conversion at higher enzyme loading (0.5-1 mgenzyme gPET -1 ).More importantly, the enzyme activity would be severely reduced at high product concentrations in an industrially relevant scale.Alongside the solids increase, a decrease in the final yield is often observed in enzymatic hydrolysis of biomass, which is generally known as the highsolids effect.The overall conversion of cellulose decreased from ~65% to 40% as solids loadings increased from <100 g kg -1 to 175 g kg -1 (Biomass.Bioenergy 2013, 56, 526-544).This has been inconclusively attributed to water constraint or the inhibition of enzymes by the high concentration of its products (Biotechnol Biofuels. 2020, 13, 58).
In our comparative analysis with the well-known engineered PET-degrading enzymes, their enzymatic activity under industrially-relevant conditions (high substrate loadings, elevated enzyme concentrations) failed to exceed that of LCC ICCG , although they demonstrated remarkable degradation capabilities in their reported conditions (Figure 2).Hence, despite the progressive strides in PET enzyme engineering, the efficiency of PET enzymatic hydrolysis under industrial conditions remains stagnant.
Concurring with the reviewer's astute observation, we recognize that this manuscript could benefit from an infusion of more scientifically robust content.We have added a more in-depth analysis of the structural features and the heterogeneous catalytic properties of TurboPETase that may explain its improved performance, as well as analysis of the catalytic performance of TurboPETase for different substrates (high crystallinity PET, soluble small molecule substrates).We sincerely hope that our revised manuscript could meet the publication standards of Nature Communications.Most importantly, we wish to convey to the wider bio-scientific community the significance of assessing enzymatic depolymerization efficiency under conditions pertinent to industry, ensuring the real-world applicability of subsequent enzyme engineering initiatives.
Comment 2: The work is more technical than scientific.The authors develop a tool for recycling PET, which they show that works well, but dont explain why it works well.The proposed increased flexibility of the active site may turn true.Still, more profound and solid studies are needed to explain the origin and rationale of the increase in enzyme performance.Such rationale is what distinguishes high-level science from technical work because it makes us understand better how nature works and inspires others in the field about ways of reaching similar goals, supporting the process of the scientific field in general.Therefore, I advise the authors to focus more seriously on explaining why the enzyme performance increased and present solid proof for their hypotheses.Otherwise the work leads to technical progress but not scientific advances.
Response: Thanks for the valuable comment.In our revised manuscript, we incorporated a series of new experiments to elucidate the potential factors contributing to the enhanced performance of TurboPETase.Firstly, we conducted a kinetic comparison for soluble substrates (MHET and pNPB).
The catalytic efficiency of TurboPETase towards MHET showed only a modest enhancement relative to PET (with a 32% increase).This observation prompted us to consider alternative factors, possibly an increased adsorption to the surface, as a potential contribution to the amplified degradation efficiency of PET.We subsequently measured of free enzyme concentrations, Efree, and converted it to substrate coverage, = (Etot -Efree)/SPET to calculated the adsorption of enzymes to PET surface.Intriguingly, upon reaching saturation, the enzymes, including TurboPETase, manifested similar maximum adsorption capacities ( max ), underscoring a consistent overall binding potential across the enzymes.Since non-specific adsorption accounts for a considerable proportion of the total adsorption sites, comparisons of these enzymes could be expanded by the consideration of inverse Michaelis Menten parameters, which can reflect the binding capability to the specific attack sites of the PET surface.The inv KM values revealed marginal differences between TurboPETase, BhrPETase, and LCC ICCG , whereas the inv Vmax/ mass S0 of TurboPETase exhibited a 2.3fold enhancement.Given that no substantial differences in inv K M and max values were observed among the enzymes, the elevated inv Vmax/ mass S0 values may imply a broadened targeting of TurboPETase towards specific attack sites when the enzymes maintained a stable overall adsorption level.Detailed discussion can be found in page 7-8, lines 233-276, Additionally, we conducted a kinetic comparison for soluble substrates (MHET and pNPB) as detailed in Supplementary Fig. S7 and Supplementary Table S6.The catalytic efficiency of TurboPETase towards MHET showed only a modest enhancement relative to PET (with a 32% increase), suggesting that there may be other factors, potentially increased adsorption to the surface, contributing to the amplified degradation efficiency of PET.Conversely, the slight decrement in TurboPETases kcat for pNPB, accompanied by a reduced binding affinity, inferred potential changes in the substrate binding domain, rendering it less conducive for other small molecule interactions.
Although the hydrolysis of PET could not meet the criteria for the conventional approach, the inverse Michaelis Menten model was more applicable.It should be noted that not all adsorption sites are competent for catalytic conversion, non-specific adsorption also accounts for a considerable proportion 43 .We measured free enzyme concentrations, Efree, and converted it to substrate coverage, = (Etot -Efree)/SPET to calculated the total adsorption of the enzymes to PET surface (Supplementary Fig. S8 and Supplementary Table S7).Consequently, we presumed that the enhanced depolymerization performance of TurboPETase may rely, at least in part, on the enhanced ability to attack a broader spectrum of specific attack sites that can be hydrolysed to form a productive complex.
Upon analyzing the Michaelis Menten parameters for pNPB, we found a slight decrease in TurboPETases k cat accompanied by a reduced binding affinity.This suggests potential changes in the substrate binding domain of TurboPETase, rendering it less conducive for other small molecule interactions.Therefore, we have provided a more detailed analysis of potential local structural changes and impacts on PET binding induced by the mutations.The new insights may offer a more nuanced molecular understanding of the enhanced catalytic performance of TurboPETase.Please refer to page 10-11, lines 305-329, H218 is suggested to form an intimate packing with the conserved W190 in analogous enzymes.Chen et al. found that PET hydrolytic activity could benefit from a more flexible active site in the H214S/F218I double mutant (corresponds to H218S/F222I in BhrPETase) 35 .In the present study, MD simulations of the apo form of TurboPETase revealed an expanded rotational freedom of W190 endowed by the H218S/F222I mutation (Supplementary Fig. S11), which is consistent with the observation of diverse conformations of the corresponding W156 of IsPETase 35 .When binding to the PET, the wobbling of W190 is curtailed and anchored by the -interactions with the PET substrate.This flexibility of the PET binding cleft was further enhanced by the synergistic interactions conferred by the addition of W104L and F243T, as revealed by the C root-mean-square fluctuation (RMSF) results (Fig. 3C).W104 is previously reported to pack against the adjacent P248 to stabilize the P248-situated 8-6 loop 34 .In the redesigned TurboPETase, the relinquishment of this interaction by leucine substitution may engender increased conformational malleability within the loop region (N246-A250), as demonstrated by the largely reduced cross-correlation of these regions (Supplementary Fig. S12).For another substitution F243T in the PET binding cleft, the steric profile of F243 appears to dictate a more peripheral binding locus for PET.Yet, its mutation to threonine, armed with a less pronounced steric feature, may release the space for PET binding with a more-flexible state.More importantly, without the steric profile of the aromatic ring, T243 may beckon PET deeper into the cleft, drawing the substrates labile carbonyl closer to the catalytic serine, with the interstitial distances contracting from 4.88 ± 0.51 Å to 4.15 ± 0.37 Å (Supplementary Fig. S13).Concurrently, the enhanced flexibility might compromise the protein's stability, which is consistent with the observed decrease in the melting temperatures of the single point mutations.
Synthesizing our structural analysis with the kinetic data, we postulated that the greatly increased flexibility along the PET-binding groove may provide more space to accommodate a variety of attack conformations through dynamic binding to enhance the ability to attack a broader spectrum of specific attack sites.We earnestly hope that our revised manuscript offers more insights into the potential mechanisms underlying the improved depolymerization performance of TurboPETase, and now strikes a balance between technical progression and scientific advancement.

Reviewer 2:
Comment 1: Because the wt BhrPETase is 94% identical to the wt LCC, the resulting mutant TurboPETase with 8 single mutations is still very similar to the reference LCC ICCG enzyme.Since a wider audience may not be aware of this high similarity due to the nomenclatures used to call these enzymes, which is also not emphasized in the revised manuscript, I believe it is reasonable to include a figure in the main text (e.g., an extended form of Fig. S5 to include structural and sequence information also about LCC and ICCG variant instead of only being shown in the supplementary information) to illustrate the sequence alignment of these four enzyme variants as well as a structural alignment based on co-crystallized ligand (e.g., PDB ID 7VVE) to highlight the variable positions that have been mutated in this study.Possibly also the few additional ones that were left out of the mutagenesis but are still close to the binding groove.Response: Thanks for the reviewers comment.We recognize the importance of providing the sequence information for these enzymes, as it would offer readers a clearer insight into their interrelationships.In the revised manuscript, we have added the structural comparison of TurboPETase with its counterparts LCC ICCG and BhrPETase as depicted in Figure 2. The local structure of the mutation site has been magnified to ensure readers can distinctly perceive the location of the mutation.Given the limitations of incorporating extensive content into the main figure, we added the sequence alignment of these enzymes to the Supplementary information.Additionally, weve elucidated the relationship between these enzymes.Kindly refer to page 3, lines 97-100 in the revised manuscript, BhrPETase shares a high sequence identity of 94% with LCC, whereas LCC ICCG represents a variant of LCC characterized by four amino acid substituents (F243I/D238C/S283C/Y127G).
Comment 2: Although the authors could have revised the rationale for using the language model and other computational tools (yellow-highlighted text on page 3 and the supplementary discussion on page 11 of the supplementary information file), the utility of these models appears to be quite limited to merely identifying two mutagenesis target positions which are obviously not unique: W104 has been reported by Zeng et al. (ref. no. 30 regarding protein engineering of the highly similar LCC ICCG benchmark enzyme) for its role in the interaction with the substrate; F243 (and its equivalent position) has been repeatedly identified as a mutagenesis hot spot for LCC ICCG (e.g., by Tournier et al. in Nature and many follow-up researches published afterwards) or other highly homologous PETases (e.g.,ref. no. 32) .Therefore, the novelty, usefulness and effectiveness of this method should not be overemphasized as described in the current version of the manuscript, e.g., the statement in the rebuttal letter The results showed that W104 and F243 are difficult to predict by simple sequences alignment, demonstrating the advantages of using machine learning to uncover hidden information regarding the improvement of polymer degradation.Accordingly, the limitation of this method should be stated.In contrast, the GRAPE strategy, despite its limited novelty, has suggested four of the eight useful residue substitutions in TurboPETase, which appears to be more effective for universal use.In this context, as described in lines 267 and 268, the A251C/A281C double mutations should form a disulfide bridge that is distinct from those introduced to LCC-ICCG and many other PETases at an alternative position.I am unable to find experimental evidence that the A251C/A281C disulfide bridge is actually formed.This must be demonstrated using structural biology techniques or biochemical methods.Response: Thanks for the reviewers comment.Indeed, as noted by the reviewer, position F243 represents a mutated site in LCC ICCG as reported by Tournier et al.However, W104 position has not been reported to enhance the depolymerization performance so far.In reference 30, Zeng et al. suggested that W104 has packing interactions with its mutated residue N248P thereby enhancing the protein thermostability, rather than an interaction with the substrate to promote its depolymerization properties.Nonetheless, we understand the reviewers concerns about the usefulness and effectiveness of the Transformer model.We have revised our statement regarding this method, discussing the limitations and potential future optimization directions for this approach.Please refer to the revised Supplementary Discussion, These results demonstrated the difficulty in predicting W104 and F243 using bioinformatics methods.Recent successes in enzyme design guided by statistical models or neural networks informed by protein family data or multiple sequence alignment (MSA) have highlighted the rich information encoded in the sequence space of natural enzymes associated with certain functionalities 11-13 .
While the Transformer model offers some promising insights, it is not devoid of limitations.One notable concern is its potential inadequacy for orphan enzymes that lack a substantial number of homologous sequences.Secondly, it is challenging to distinguish functionally relevant signals from noise in diverse sequences.Given the increased variability in the N-terminal and C-terminal sequences compared to the core of the protein, our method had a bias toward more variable at N-and C-terminals.Manual removal of such regions is necessary.Additionally, the current model does not consider the structural information, which has been demonstrated as valuable in other engineering efforts 14-15 .Incorporating such information presents a potential avenue for refining our approach.Furthermore, given the extensive research efforts directed towards the engineering of PET hydrolases, integrating available experimental measurement data could further enhance the robustness and accuracy of the algorithm in subsequent iterations.
For disulfide bond, we have confirmed the disulfide bond formation using Ellmans reagent, which has also been used by Pfaff et al. (ACS Catal. 2022, 12, 9790-9800).Please refer to Supplementary Figure S10.
Comment 3: In Figure 2, the authors could present new experimental data for comparing different PETases under optimal buffer and temperature conditions.In general, lower ionic strengths and buffer concentrations are used in the new experiments, which are also related to the final depolymerization experiments with high substrate solid loading.Nonetheless, based on the information provided in the supplementary information, 1.7(1) and 1.10, the comparison of the activity of various mutants (Fig. 1D) and kinetic analysis (Figure 3 and Table 1) still only included data collected with 1 M potassium buffer, which can significantly (rather positively) influence enzyme activity, stability, and adsorption behavior to the substrate (, and this is widely accepted by scientific communities).This may render the mutant ranking invalid and result in incomparable kinetic properties to the degradation experiments depicted in Figures 2 and 4. The ideal solution is to repeat the kinetic analysis at lower buffer concentrations and, at the very least, probe several mutants shown in Fig. 1D to ensure that the use of 1 M buffer has no (negative) effect on their ranking in enzyme activity comparison.Furthermore, neither the main text nor the supplementary information explains how and in which buffer the Tm of individual mutants was determined.As previously stated, if 1 M buffer was used, certain mutants could be sufficiently thermoactivated and stabilized.Therefore, the superior mutant TurboPETase deduced based on the data in Fig. 1D and the supplementary tables may not be the ideal one to be used in a buffer-free condition (Fig. 4B).Comparing the old datasets (which have been moved to Fig. S4) with the new datasets in Fig. 2, it was also observed that the use of 1 M buffer can improve the degradation performance of TurboPETase by approximately 4-fold (at 50°C) or 7-fold (at 65°C) at similar enzyme to substate ratios but low solid loading levels.From an opposing perspective, have the authors considered confirming this benefit of using a high buffer concentration for large-scale, high-solid-loading experiments such as those depicted in Figure 4? For a future industrial application in the real world, a high buffer concentration will undoubtedly raise numerous (such as cost-related) questions.However, it would be of great scientific interest to determine if a 1 M buffer will further increase the degradation efficiency of TurboPETase and, if so, to discuss whether it would be preferable to use a low salt concentration at the expense of drastically reducing the degradation efficiency.Response: Thanks for the reviewers comment.The Tm was determined in the enzyme storage buffer (50 mM Na2HPO4 and 100 mM NaCl, pH 7.5).We have added the descriptions in the methods section in the supplementary materials.Please refer to section 1.9 Determination of apparent melting temperatures and PET crystallinity in the revised Supplementary materials, A fluorescence- based thermal stability assay was used to determine apparent melting temperatures.Protein solution (20 µL) in the buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.5) was mixed with 5 µL 100-fold diluted SYPRO Orange dye (Molecular Probes, Life Technologies, USA) in a thin-walled 96-well PCR plate.The plate was sealed with opticalquality sealing tape and heated in a CFX 96 real-time polymerase chain reaction (PCR) system (BioRad, Hercules, CA, USA) from 25 to 100 °C at a heating rate of 1.4 °C/min.Fluorescence changes were monitored with a chargecoupled device (CCD) camera.The wavelengths for excitation and emission were 490 and 575 nm, respectively.The parameters were set following a previously reported procedure 3 .
As the reviewer suggests, we compared the depolymerization performance of the 12 variants based on the M6 scaffold (Figure R1) and executed additional experiments for kinetic analysis employing 100 mM KH2PO4-NaOH buffer.As shown in Figure R1, TurboPETase was still the most active variant in respect to other variants at lower buffer concentrations.The new kinetic results also demonstrated comparable inv Km among the evaluated enzymes and improved inv Vmax/ mass S0 values for TurboPETase, which are similar to the results in the original manuscript.Thus, the main conclusion has not changed.We have redrawn the figures with the new data, please refer to Figures 3A and B in the revised manuscript.
Furthermore, we agree with the reviewer that higher phosphate concentrations (up to 1 M) may largely promote the PET hydrolysis activity for certain enzymes, such as PES-H1 (ACS Catal.2022, 12, 9790-9800) and M6-W104G/F243T mutant in this study.For TurboPETase, however, the performance enhancement under these conditions appears to be more restrained.A 1M phosphate concentration could improve the hydrolysis activity by a modest ~10% at an enzyme loading of 2 mgemzyme gPET -1 at 65 °C.This increment is slightly more pronounced, around 34%, at lower enzyme concentrations (0.3 mgemzyme gPET -1 ) at 65 °C.We speculate that the 4-fold (at 50°C) or 7-fold (at 65°C) increase inferred by the reviewer may stem from the comparison of degradation performance under varying enzyme concentrations and substrate loadings.Additionally, the disparity could also be attributed to the comparison with LCC.The activity of LCC is inhibited under conditions with 1 M phosphate buffer concentration, thereby widening the disparity in catalytic efficiency when com pared to TurboPETase.To facilitate a more transparent comparison, we listed the degradation effici ency of TurboPETase under different conditions in the appended table.These data can also be foun d in the in the source data submitted to both [another Nature journal] and Nature Communication s.Considering the marginal influence of buffer concentration on TurboPETases degradati on efficiency at high enzyme concentrations, coupled with the considerable economic a nd environmental costs associated with phosphate removal in industrial reaction conditions, we advocate for preserving the current conditions for large-scale reactions.
Table R1.PET monomers released from hydrolysing Gf-PET films with TurboPETase at temperatures ranging from 50 to 65 °C for 3 h, using solids loading of 30 g kg -1 .All measurements were conducted in triplicate (n = 3).Comments 4-6: The depolymerization performance with TurboPETase, as shown in Figure 4, and the maximum production rate described in line 289, are unquestionably the highlights of this study.However, based on the polymer property data available thus far, the PET substrate used in this study has a crystallinity of 10.9%, which is significantly lower than the 14.6% used by Tournier et al ( 2020) with LCC ICCG.The final crystallinity of residual PET materials at 65°C as a result of physical aging and degradation-induced crystallization is in the range of 12.3-12.8%,which is still significantly lower than the starting material used by Tournier et al.As a result, I believe that the remarkable degradation performance should rather be attributed to the more "heavily amorphized" waste PET in comparison to the less amorphized materials used by Tournier et al.Furthermore, ref.
32. indicated that the molecular weights of the pretreated PET waste may influence their degradability; thus, the authors should provide GPC analysis of their pristine PET waste, those after extrusion, after micronization, and after enzymatic degradation individually for a better understanding of the correlation between polymer property and degradability.
The inferior performance of LCC ICCG at 65°C shown in Fig. 4 appears plausible to me, consistent with the data shown in Fig. 2 and also by Tournier et al., who stated that LCC ICCG should perform similarly to wt LCC at 65°C.However, the marginally better-performed degradation curve with LCC ICCG at 72°C with a similar shape to that determined at 65°C until at least 14 h did not appear to be reasonable.This is in stark contrast to previous studies such as those by Tournier et al All previous studies clearly demonstrated a significantly higher (~1.5 to 2-fold) degradation activity of LCC ICCG at temperatures ranging from 72-74°C compared to those at 65°C to 66°C.Given the lower crystallinity of the substrate used in this study (10.9%), the degradation performance with LCC ICCG at 72°C did not appear to be reliable or reproducible (i.e. should be much higher).Furthermore, as shown in Fig. S1B, the crystallinity of the PET substrate at 72°C exceeded 20% after 4 hours of reaction; however, it did not appear that the LCC-ICCG reaction rates were significantly reduced before 12 hours.The degradation curve at 72°C behaved in some ways like a "parallel" curve with the one determined at 65°C, where the crystallinity of the PET substrate continuously maintained less than 15% in correlation to a high degradability.This makes absolutely no sense to me.
I observed that the degradation curves in Fig. 4A and Fig. S6 were calculated using HPLC analysis, whereas the one depicted in Fig. 4B was determined using only NaOH consumption.I believe that these data ought to be better validated, similar to those presented by Tournier et al. in their extended Response: Thank you for the insightful comments.We deeply value the reviewers expertise and have taken the observations seriously, ensuring our data is not only accurate but also aligns with the broader scientific consensus.
According to the reviewers suggestion, we have revised the depolymerization data based on the combinatorial data obtained by NaOH consumption, HPLC analysis, and the weight loss determinations.Striving for the consistency with the work of Tournier et al., we recalibrated the initial rate, specifically basing it on NaOH consumption.For a detailed data, please refer to the Supplementary Table S9.The following is the content from Supplementary Table S9.Table S9.Enzymatic depolymerization of pretreated PET powder in the bioreactors.

Enzymatic treatment scale
Enzyme, temperature and time
Notably, the depolymerization values derived from NaOH consumption appeared to be somewhat lower than that calculated from HPLC, which can be attributed to the residual unhydrolyzed MHET present in the solvent.We have labeled the data source in the revised manuscript, please refer to page 11, lines 343-352, TurboPETase achieved nearly complete depolymerization (98.2%, calculated from the HPLC data) of PcPET wastes in 8 h (Fig. 4A),In contrast, LCC ICCG required 16 hours to reach 97.7% depolymerization (calculated from the HPLC data) at 65 °C, At the previously reported optimal reaction temperature of 72 °C, LCC ICCG reached its maximal conversion of 92.5% (calculated from the HPLC data) over 12 h, and no further increase was obtained after prolonged reaction time due to the higher deformability of PET chains.And page 13, lines 386-388, Despite the decreased hydrolytic efficiency, the approximately 98% depolymerization (98.9% calculated from the HPLC data, 98.4% calculated from the consumed NaOH and 97.4% calculated from the weight loss, as listed in Supplementary Table S9) achieved within 8 hours during the scaled-up reaction (Fig. 4D) makes pilotscale production feasible.
Upon further analysis of our recalibrated data, the initial rate for LCC ICCG at 72°C was 1.6 times that at 65°C.This observation resonates with the reviewers point and previously documented studies that underscore LCC ICCG s augmented degradation activity (by around 1.5 to 2-fold at elevated temperatures (72-74°C) compared to 65°C.Furthermore, we also extracted the remaining PET (~20% crystallinity) from reaction solvent after 4 h reaction.As per the reviewer's request, GPC measurements of the plastic were also provided (Table R2).Following thorough washing and desiccation, we added fresh LCC ICCG for further degradation.The results showed a comparable hydrolysis activity of LCC ICCG on 10.7% crystallinity PET at 65°C to that on 20% crystallinity PET at 72°C (Table R3).This suggests a parallel degradation trajectory of LCC ICCG at 72°C to that at 65°C between 4-8 hours of reaction.Pfaff et al. demonstrated that LCC ICCG hydrolyzed shorter polymers more efficiently (ACS Catal. 2022, 12, 9790-9800).We speculate that due to the much lower Mn of the pretreated PcPET respect to other PET materials, LCC ICCG didn't exhibit a rapid decline in degradation rate during the reaction at 72°C when the PET crystallinity reached 20%.However, when the crystallinity increased to 33%, the substantial reduction in the amorphous regions hindered further catalysis.We acknowledge the crystallinity discrepancy (10.9% in our study vs. 14.6% by Tournier et al.).Regrettably, employing the same techniques, we couldn't achieve samples with comparable crystallinity.To mitigate this, we assessed TurboPETase's degradation performance on readily accessible crushed PET powders sourced from Coca-Cola bottles (with a crystallinity of 27.6%), ensuring that a broad range of researchers could replicate our findings.As depicted in Figure R2, TurboPETase's degradation efficiency for high-crystallinity plastic is 1.5 times that of LCC ICCG (Figure R2), demonstrating that TurboPETase's augmented catalytic prowess isn't narrowly tailored to plastics with low crystallinity but is robustly applicable to high-crystallinity PET as well.
Comment 7: I am unable to fully comprehend the statement between lines 299 and 301, which I find to be quite confusing.I believe it is necessary for the authors to provide data and more thorough descriptions of the "water constraint" and "supersaturated TPA" concentration profiles under various reaction conditions.Response: We apologize for the confusion caused by this description.To prevent similar queries from other readers and considering that this observation does not significantly contribute to the main narrative of the article, we have removed this statement from our original submission to Nature Communications.
Comment  2E).Under their reported reaction conditions, the reaction rates of LCC ICCG , HotPETase, and FastPETase were 1.8-, 4.9-, and 1.0-fold lower, respectively, than that of TurboPETase.Given the reported enhancements of PES-H1 L92F/Q94Y in 1 M KH2PO4-NaOH buffer, we also compared TurboPETase with PES-H1 L92F/Q94Y under elevated buffer concentration, with TurboPETase still outpacing, yielding up to 2.5 times the degradation products of PES-H1 L92F/Q94Y at 65 °C (Supplementary Fig. S6).Nonetheless, we agree with the reviewer's comments and have revised our manuscript to The redesigned variant, TurboPETase, outperformed other well-known PET hydrolases. in the abstract and also in page 2, lines 70-78, The redesigned variant (TurboPETase) derived from this campaign outperformed the most efficient PET hydrolases currently recognized in the field (LCC 21 , LCCICCG 17 , ICCG I6M22 , BhrPETase 23 , FastPETase 24 , HotPETase 18 , DepoPETase 25 , CaPETase M926 , and PES-H1 L92F/Q94Y27 ) over a range of temperatures (50 °C-65 °C).The extraordinary degradation performance afforded by TurboPETase allowed nearly complete depolymerization of postconsumer PET bottles in 8 h at a high industrially relevant substrate loading of 200 g kg -1 , with a maximum production rate of 61.3 ghydrolyzed PET L -1 h -1 , addressing the challenge regarding residual nonbiodegradable PET waste.
Once again, we extend our gratitude for your insightful comments, which have undeniably enriched the depth and rigor of our study.Specifically, your guidance has allowed our data and analyses to align more closely with the standards of our field, bolstering the quality and credibility of our work.
We are truly honored to receive such constructive and professional feedback.Thank you for your substantial contribution to enhancing the quality of our paper.

Reviewer 3:
Comment 1: The manuscript entitled Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading provides some interesting information related to enzyme development.However, given the high number of articles published in this and other journals related to incremental improvements of PET hydrolyzing activity of various enzymes, the novelty of this article seems to be very limited.The authors tried to compare the activity of their enzyme to some recently published PET hydrolyzing enzymes while it is not clear how e.g.different reaction conditions or enzyme thermostabilities may affect the comparison in figure 4. Apart from decomposition of PET, a lot of other parameters would be interesting to be included in a more mechanistic comparison of different PET hydrolyzing enzymes, such as sorption characteristics, activity on small water soluble (model) substrates, release of oligomers and activity thereon, specificity for other aromatic / aliphatic polyesters, potential synergies with other enzymes etc.
Response: We appreciate the meticulous feedback provided by the reviewer, highlighting the need for a more comprehensive analysis on enzyme development for PET hydrolysis.As the reviewer suggests, we have added a more in-depth analysis of the catalytic properties of heterogeneous systems, the catalytic performance of TurboPETase for different substrates, specificity for other aromatic polyesters (PEN and PBT), and potential synergies with the recently reported BHETase.
Firstly, we conducted a kinetic comparison for soluble substrates (MHET and pNPB).The catalytic efficiency of TurboPETase towards MHET showed only a modest enhancement relative to PET.This observation prompted us to consider alternative factors, possibly an increased adsorption to the surface, as a potential contribution to the amplified degradation efficiency of PET.We subsequently measured of free enzyme concentrations, Efree, and converted it to substrate coverage, = (Etot -Efree)/SPET to calculated the adsorption of enzymes to PET surface.Intriguingly, upon reaching saturation, the enzymes, including TurboPETase, manifested similar maximum adsorption capacities ( max ), underscoring a consistent overall binding potential across the enzymes.Since nonspecific adsorption accounts for a considerable proportion of the total adsorption sites, comparisons of these enzymes could be expanded by the consideration of inverse Michaelis Menten parameters, which can reflect the binding capability to the specific attack sites of the PET surface.The inv KM values revealed marginal differences between the enzymes, whereas the inv Vmax/ mass S0 of TurboPETase exhibited a 2.1-fold enhancement, which may imply a broadened targeting of TurboPETase towards specific attack sites when the enzymes maintained a stable overall adsorption level.Detailed discussion can be found in page 7-8, lines 233-276, Additionally, we conducted a kinetic comparison for soluble substrates (MHET and pNPB) as detailed in Supplementary Fig. S7 and Supplementary Table S6.The catalytic efficiency of TurboPETase towards MHET showed only a modest enhancement relative to PET (with a 32% increase), suggesting that there may be other factors, potentially increased adsorption to the surface, contributing to the amplified degradation efficiency of PET.Conversely, the slight decrement in TurboPETases kcat for pNPB, accompanied by a reduced binding affinity, inferred potential changes in the substrate binding domain, rendering it less conducive for other small molecule interactions.
Although the hydrolysis of PET could not meet the criteria for the conventional approach, the inverse Michaelis Menten model was more applicable.It should be noted that not all adsorption sites are competent for catalytic conversion, non-specific adsorption also accounts for a considerable proportion 43 .We measured free enzyme concentrations, Efree, and converted it to substrate coverage, = (Etot -Efree)/SPET to calculated the total adsorption of the enzymes to PET surface (Supplementary Fig. S8 and Supplementary Table S7).Consequently, we presumed that the enhanced depolymerization performance of TurboPETase may rely, at least in part, on the enhanced ability to attack a broader spectrum of specific attack sites that can be hydrolysed to form a productive complex.
Upon analyzing the Michaelis Menten parameters for pNPB, we found a slight decrease in TurboPETases kcat accompanied by a reduced binding affinity.This suggests potential changes in the substrate binding domain of TurboPETase, rendering it less conducive for other small molecule interactions.Therefore, we have provided a more detailed analysis of potential local structural changes and impacts on PET binding induced by the mutations.The new insights may offer a more nuanced molecular understanding of the enhanced catalytic performance of TurboPETase.Please refer to page 10-11, lines 305-329, H218 is suggested to form an intimate packing with the conserved W190 in analogous enzymes.Chen et al. found that PET hydrolytic activity could benefit from a more flexible active site in the H214S/F218I double mutant (corresponds to H218S/F222I in BhrPETase) 35 .In the present study, MD simulations of the apo form of TurboPETase revealed an expanded rotational freedom of W190 endowed by the H218S/F222I mutation (Supplementary Fig. S11), which is consistent with the observation of diverse conformations of the corresponding W156 of IsPETase 35 .When binding to the PET, the wobbling of W190 is curtailed and anchored by the -interactions with the PET substrate.This flexibility of the PET binding cleft was further enhanced by the synergistic interactions conferred by the addition of W104L and F243T, as revealed by the C root-mean-square fluctuation (RMSF) results (Fig. 3C).W104 is previously reported to pack against the adjacent P248 to stabilize the P248-situated 8-6 loop 34 .In the redesigned TurboPETase, the relinquishment of this interaction by leucine substitution may engender increased conformational malleability within the loop region (N246-A250), as demonstrated by the largely reduced cross-correlation of these regions (Supplementary Fig. S12).For another substitution F243T in the PET binding cleft, the steric profile of F243 appears to dictate a more peripheral binding locus for PET.Yet, its mutation to threonine, armed with a less pronounced steric feature, may release the space for PET binding with a more-flexible state.More importantly, without the steric profile of the aromatic ring, T243 may beckon PET deeper into the cleft, drawing the substrates labile carbonyl closer to the catalytic serine, with the interstitial distances contracting from 4.88±0.51Å to 4.15±0.37Å (Supplementary Fig. S13).Concurrently, the enhanced flexibility might compromise the protein's stability, which is consistent with the observed decrease in the melting temperatures of the single point mutations.Synthesizing our structural analysis with the kinetic data, we postulated that the greatly increased flexibility along the PET-binding groove may provide more space to accommodate a variety of attack conformations through dynamic binding, which may be crucial for the formation of catalytically competent complexes on different surface structures (Fig. 3D).
To extend the potential applications of TurboPETase, we also examined the use of this enzyme for the degradation of another semiaromatic polyesters, PBT (Supplementary Figure S15).
Compared to PET, PBT has slightly lower strength and rigidity, slightly better impact resistance, and a slightly lower glass transition temperature.Even though 65°C surpasses the glass transition temperature of PBT (Tg ranging between 37 to 55°C), thus substantially enhancing the mobility of PBT polymer chains, all of the examined enzymes exhibited substantially reduced degradation efficiency toward PBT films at 65 °C with respect to the degradation of PET.Specifically, TurboPETase yielded higher amounts of hydrolytic products (62.6 M) than both BhrPETase (27.5 M) and LCC ICCG (47.1 M).These results demonstrated the challenge for the active sites of current PET-degrading enzymes to efficiently binding with the extended aliphatic chains in PBT, compared to those in PET.We have added the discussion in the revised Supplementary Discussion, please refer to the section 2.2 Depolymerization of Polybutylene terephthalate (PBT) in the Supplementary materials, We examined the use of TurboPETase for the degradation of another semiaromatic polyester, PBT (Fig. S15).Compared to PET, PBT has slightly lower strength and rigidity, slightly better impact resistance, and a slightly lower glass transition temperature.Even though 65°C surpasses the glass transition temperature of PBT (Tg ranging between 37 to 55°C 16 ), thus substantially enhancing the mobility of PBT polymer chains, all of the examined enzymes exhibited substantially reduced degradation efficiency toward PBT films at 65 °C with respect to the degradation of PET.Thus, dedicated efforts in enzyme discovery or tailored engineering are still needed for further improving the depolymerization of new classes of semiaromatic polyesters.
We further explored the potential of TurboPETase by coupling it with the recently reported BHET hydrolyzing enzyme, BHETase (Nat.Commun. 2023, 14, 4169), in a dual-enzyme system.At a low substrate loading (2 g kg -1 ), this combination substantially elevated the overall yield of the products (sum of BHET, MHET and TPA), relative to the singular use of TurboPETase.However, an intriguing observation emerged at an elevated PET loading of 30 g kg -1 .TurboPETase alone surpassed the yields from most enzyme ratios in the dual-enzyme system, the only exception being the 0.5 mgTurboPETase/gPET:0.1 mgBHETase/gPET ratio.This observed trend echoes a previously report wherein binding modules were added to LCC YCCG (Chem Catal. 2022, 2, 26442657).Owing to the ability of fusion enzymes to hydrolyze BHET, the resultant fusion enzymes exhibited superior performance at low substrate loadings (< 3 wt% PET).However, as the PET loading intensified (up to 10-20 wt%), the fusion enzymes TrCBM1, TtCBM10, and StCBM64 show no sustained advantage over the LCC YCCG domain alone over the enzyme concentration range 50 nM to 1 mM.They proposed a potential explanation suggesting that the increased solids loading increases the frequency of enzyme-substrate interactions, thereby accelerating PET hydrolysis to such an extent that the presence of the other enzyme no longer provides any additional benefit.In the current investigation, only amorphous PET materials were evaluated.Hence, subsequent studies could explore the behavior of the dual-enzyme system across PET substrates possessing varied physical morphologies, taking into account factors like crystallinity, accessible surface area, and chemical purity.However, such an exploration falls outside the primary focus of this manuscript.We have added the discussion in the revised Supplementary Discussion, please refer to the section 2.3 Depolymerization of Gf-PET films with a dual-enzyme system in the Supplementary materials, We further explored the coupling of TurboPETase with the recently reported BHET hydrolyzing enzyme, BHETase 17 , in a dual-enzyme system.At a low substrate loading (2 g kg -1 ), the dual-enzyme system effectively doubled the overall yield of the products (sum of BHET, MHET, and TPA), relative to the singular use of TurboPETase (Fig. S16).However, an intriguing observation emerged at an elevated PET loading of 30 g kg -1 .TurboPETase alone surpassed the yields from most enzyme ratios in the dualenzyme system, the only exception being the 0.5 mgTurboPETase/gPET:0.1 mgBHETase/gPET ratio.This observed trend echoes a previously report wherein binding modules were added to LCC YCCG 18.The fusion enzymes exhibited superior performance at low substrate loadings (< 3 wt% PET).However, as the PET loading intensified (up to 10-20 wt%), they show no sustained advantage over the LCC YCCG domain alone over the enzyme concentration range 50 nM to 1 mM.Nonetheless, in the current investigation, only amorphous PET materials were evaluated.Additional investigations could be conducted to explore the behavior of the dual-enzyme system across PET substrates possessing varied physical morphologies, taking into account factors like crystallinity, accessible surface area, and chemical purity.
Based on these results and the previously reported observations, wed like to further emphasize the importance of standardizing the reaction conditions to an industrially relevant setting for comparing the performance of various enzymes.
As in the response to reviewer 1, we have witnessed a number of success in the engineering of PET hydrolases over the last 3 years, gearing towards potential applications in industrial plastic recycling processes.However, a majority of works aiming at enhancing enzyme performance are performed in small-scale reactions, often at low enzyme and substrate concentrations.In heterogeneous catalysis reactions, different enzymes may exhibit dramatically distinct catalytic efficiencies at various enzyme and substrate concentrations, as in the case of cellulases (ACS Catal. 2017, 7, 4904-4914;Biotechnol Biofuels. 2020, 13, 58).For instance, Erickson et al. demonstrated that IsPETase showed similar hydrolytic conversion compared to its variant IsPETase W159H/S238F at low enzyme loading (0.5-1 mgenzyme gPET -1 ), but approximately 2-fold lower conversion at higher enzyme loading (0.5-1 mgenzyme gPET -1 ).More importantly, the enzyme activity would be severely reduced at high product concentrations in an industrially relevant scale.Alongside the solids increase, a decrease in the final yield is often observed in enzymatic hydrolysis of biomass, which is generally known as the high-solids effect.The overall conversion of cellulose decreased from ~65% to 40% as solids loadings increased from <100 g kg -1 to 175 g kg -1 (Biomass.Bioenergy 2013, 56, 526-544).This has been inconclusively attributed to water constraint or the inhibition of enzymes by the high concentration of its products (Biotechnol Biofuels. 2020, 13, 58).In our comparative analysis with the well-known engineered PET-degrading enzymes, their enzymatic activity under industrially-relevant conditions (high substrate loadings, elevated enzyme concentrations) failed to exceed that of LCC ICCG , although they demonstrated remarkable degradation capabilities in their reported conditions (Figure 2).Hence, despite the progressive strides in PET enzyme engineering, the efficiency of PET enzymatic hydrolysis under industrial conditions remains stagnant.We have added this discussion in the revised manuscript, please refer to page 13, lines 398-405, However, the material slurry exhibits a high apparent viscosity due to the high solids loading, leading to limited mass and heat transfer, which reduces the efficiency of enzymes in the early stages of hydrolysis.More importantly, increasing the solids loading to industrially relevant levels would lower the depolymerization yield due to the inhibition by high product concentrations 16 .Hence, mere thermalstability of the enzyme may not suffice for industrial PET degradation.Multiple factors interplay, influencing enzyme efficacy in real-world scenarios.
We sincerely hope that our revised manuscript could meet the publication standards of Nature Communications.Most importantly, we wish to convey to the wider bio-scientific community the significance of assessing enzymatic depolymerization efficiency under conditions pertinent to industry.This perspective, we feel, is pivotal for advancing PETase engineering endeavors for the real-world applicability.
Once again, we appreciate the reviewers' time and effort given to our manuscript and are very grateful for their invaluable comments that have led to the significant improvement of the presentation of our work.
I appreciate the serious effort and additional experiments made to address my criticisms of the original version of the manuscript.The manuscript is much deeper and more scientific, as the authors elucidated the underlying reasons for the improved performance of TurboPETase.This opens ways for further rational optimization and provides guidelines for the community to apply similar techniques to other enzymes, increasing the broadness and appeal of the work for the community.In my opinion, the article can be published in the present form.
Reviewer #2 (Remarks to the Author): I would like to begin by expressing my appreciation for the authors' diligent efforts in conducting additional experiments to address the concerns raised in my previous review.These efforts have notably enhanced the quality of the manuscript.However, there are several aspects that I believe require further attention to ensure the manuscript's coherence and completeness.Firstly, I observed that some new data have been included only in the rebuttal letter, with references such as Table R1 and Figure R1.These data appear to be of significant value and merit inclusion in the manuscript.I recommend incorporating them partly into the Supplementary Information (partly directly into the main text), with appropriate references and discussions in the main text.This approach would provide a more integrated and comprehensive understanding of the findings.Regarding the new Figure S14 on PBT degradation and Figure S15 on dual enzyme degradation, I noticed that these figures are not sufficiently discussed (rather not mentioned at all) in the main text.Given the recent interest and publications in high-impact journals on the latter topic regarding S15, it would be beneficial to include a more thorough discussion and relevant citations in the main text if the authors decided to include Fig. S15 in the final manuscript.This would not only enhance the manuscript's relevance but also provide a more complete narrative.In addition to these points, I have identified a few specific issues that need to be addressed or clarified, possibly with the inclusion of new data, before I can fully endorse this manuscript for publication in Nature Communications: 1.) Figures R1 and Table R1 should be included in the Supplementary Information, with references and discussions in the main text.The influence of various buffers at different concentrations on the performance of PETases is a critical aspect of understanding activity differences under laboratory and industrial conditions.2.) Table R3 and Figure R2 should also be included in the Supplementary Information, with brief references and discussions in the main text.
to other PET materials, LCCICCG didn't exhibit a rapid decline in degradation rate during the reaction at 72°C when the PET crystallinity reached 20%.However, when the crystallinity increased to 33%, pivotal as it suggests that once the PET polymer chain length is reduced to a certain threshold (e.g., Mn lower than 9,198, equivalent to a degree of polymerization (DP) range of 45-47), crystallinity is no longer the sole determinant factor for enzymatic degradation.This is a significant finding, as it challenges the previously suggested threshold of less than 20% crystallinity required for enzymatic more than 20,000.This nuanced understanding aligns well with the viewpoints suggested by Pfaff et al., as well as recent papers by Guo et al. (https://pubs.acs.org/doi/full/10.1021/acscatal.1c05548,https://doi.org/10.1002/cssc.202300742),which demonstrated that heavily pretreated PET, resulting in short oligomers (DP<20), can be completely crystalline (with only trans conformers) but still remain highly degradable even by IsPETase at 30°C within a few hours.In line with this, I recommend that the authors provide data on the change of crystallinity and molecular weights of the PcPET used in the (high solid loading) degradation experiments shown in Figures 4A and B as time courses.Additionally, including time courses of % crystallinity and Mn&Mw changes under all investigated conditions in parallel to the degradation data in the same figure would greatly contribute to understanding the interplay of varying material properties essential for rapid PET degradation.4.) The new Figure 2, comparing the degradation performance of various PETases, is a valuable addition.However, I am surprised that the authors have not cited or discussed a recent publication by Carbios (https://doi.org/10.1021/acscatal.3c02922),which presents similar experiments under industrial conditions but partly with discrepant outcomes regarding the performance ranking of certain enzymes.Including this reference and discussing the discrepancies between different studies would significantly strengthen the manuscript, particularly in the context of positioning TurboPETase as a promising candidate for industrial applications.By addressing these points, the authors will significantly enhance the manuscript's contribution to the field of PET degradation, providing a more robust and comprehensive understanding of this important area of research.
Reviewer #3 (Remarks to the Author): Although the novelty of the study is still rather limited considering the large amount of papers published on PET hydrolyzing enzymes, nevertheless the authors have improved the manuscript and added a lot more data rendering it suitable for publication.
I would like to begin by expressing my appreciation for the authors' diligent efforts in conducting additional experiments to address the concerns raised in my previous review.These efforts have notably enhanced the quality of the manuscript.However, there are several aspects that I believe require further attention to ensure the manuscript's coherence and completeness.Firstly, I observed that some new data have been included only in the rebuttal letter, with references such as Table R1 and Figure R1.These data appear to be of significant value and merit inclusion in the manuscript.I recommend incorporating them partly into the Supplementary Information (partly directly into the main text), with appropriate references and discussions in the main text.This approach would provide a more integrated and comprehensive understanding of the findings.
Regarding the new Figure S14 on PBT degradation and Figure S15 on dual enzyme degradation, I noticed that these figures are not sufficiently discussed (rather not mentioned at all) in the main text.Given the recent interest and publications in high-impact journals on the latter topic regarding S15, it would be beneficial to include a more thorough discussion and relevant citations in the main text if the authors decided to include Fig. S15 in the final manuscript.This would not only enhance the manuscript's relevance but also provide a more complete narrative.
Response 1: We extend our sincere gratitude for the valuable and insightful comments throughout significantly contributed to enhancing the quality and depth of our work.Following the suggestions, we have integrated the data, previously included in the 1 st response letter, into the current revised manuscript.This includes the information in Tables R1-R3 and Figures  IsPETase and its variant towards other semiaromatic polyesters, specifically PBT, at 37 °C36 .Compared to PET, PBT has slightly lower strength and rigidity, better impact resistance, and a lower glass transition temperature (Tg), which ranges between 37 to 55 °C48 .In this study, even though 65°C surpasses the Tg of PBT, thus significantly enhancing the mobility of PBT polymer chains, all of the examined enzymes exhibited substantially reduced degradation efficiency towards PBT films at 65 °C with respect to the degradation of PET (Supplementary Fig. S15).Specifically, TurboPETase yielded higher amounts of ICCG suggested that the active sites of current PET-degrading enzymes were less efficient in binding with the extended aliphatic chains in PBT compared to PET.Consequently, dedicated efforts in enzyme discovery or tailored engineering are still needed for further improving the depolymerization of new classes of semiaromatic polyesters.
We further explored the application potential of TurboPETase by coupling it with the recently reported BHET hydrolyzing enzyme, BHETase 49 , in a dual-enzyme system.At a low substrate loading (2 g kg -1 ), the dual-enzyme system effectively doubled the overall yield of the products (sum of BHET, MHET, and TPA), relative to the singular use of TurboPETase (Supplementary Fig. S16).However, an intriguing observation emerged at an elevated PET loading of 30 g kg -1 .TurboPETase alone surpassed the yields from most enzyme ratios in the dual-enzyme system, the only exception being the 0.5 mgTurboPETase/gPET:0.1 mgBHETase/gPET ratio.This observed trend echoes a previously report wherein binding modules were added to LCC YCCG15 .The fusion enzymes exhibited superior performance at low substrate loadings (< 3 wt% PET).However, as the PET loading intensified (up to 10-20 wt%), they show no sustained advantage over the LCC YCCG domain alone.Nonetheless, in the current investigation, only amorphous PET materials were evaluated.Additional investigations could be conducted to explore the behavior of the dual-enzyme system across PET substrates possessing varied physical morphologies, taking into account factors like crystallinity, accessible surface area, and chemical purity.
In addition to these points, I have identified a few specific issues that need to be addressed or clarified, possibly with the inclusion of new data, before I can fully endorse this manuscript for publication in Nature Communications: 2.1.)Figures R1 and Table R1 should be included in the Supplementary Information, with references and discussions in the main text.The influence of various buffers at different concentrations on the performance of PETases is a critical aspect of understanding activity differences under laboratory and industrial conditions.
Response 2.1: We sincerely appreciate the revie buffers at different on PETase activity.Acknowledging the importance of this aspect, we have added a discussion in the appropriate section of the main text, please refer to pages 5-6, lines 175-To maintain the clarity and conciseness of the main text, we have included this data in the Supplementary Materials.The relevant data is now accessible in Supplementary Table S6 and Fig.
suggestions or feedback the reviewer may have.
Table R3 and Figure R2 should also be included in the Supplementary Information, with brief references and discussions in the main text.
Response 2.2 to the Supplementary Fig. S18 and Table S11.A brief discussion was also added into the main text.This discussion is seamlessly blended with our analysis of the crystallinity and molecular weight changes, providing a holistic view of the data and its implications.Please refer to page 12, lines 401- The inclusion of Table R2 in the main text is essential, particularly in conjunction pretreated PcPET respect to other PET materials, LCCICCG didn't exhibit a rapid decline in degradation rate during the reaction at 72°C when the PET crystallinity reached 20%.However, when the crystallinity increased to 33%, the substantial reduction in the amorphous regions hindered mer chain length is reduced to a certain threshold (e.g., Mn lower than 9,198, equivalent to a degree of polymerization (DP) range of 45-47), crystallinity is no longer the sole determinant factor for enzymatic degradation.This is a significant finding, as it challenges the previously suggested threshold of less than 20% with a DP greater than 100, or Mn more than 20,000.This nuanced understanding aligns well with the viewpoints suggested by Pfaff et al., as well as recent papers by Guo et al.
(https://pubs.acs.org/doi/full/10.1021/acscatal.1c05548,https://doi.org/10.1002/cssc.202300742),which demonstrated that heavily pretreated PET, resulting in short oligomers (DP<20), can completely crystalline (with only trans conformers) but still remain highly degradable even by IsPETase at 30°C within a few hours.In line with this, I recommend that the authors provide data on the change of crystallinity and molecular weights of the PcPET used in the (high solid loading) degradation experiments shown in Figures 4A and B as time courses.Additionally, including time courses of % crystallinity and Mn&Mw changes under all investigated conditions in parallel to the degradation data in the same figure would greatly contribute to understanding the interplay of varying material properties essential for rapid PET degradation.
Response 2.3: Thank you for the insightful comment and the opportunity to deepen the discussion in our manuscript.Following your suggestions, we have conducted additional analyses and expanded our discussion, particularly regarding the degradation behavior when PET crystallinity exceeds 20%.This discussion, in line with our initial speculation about the impact of the lower Mn of pretreated PcPET, can be found on page 12, lines 397-Moreover, following your suggestion, we have conducted time-course measurements of the crystallinity and molecular weights of the PcPET used in the high solid loading degradation experiments (Figure 4A).These results, which have been added to new Figure 4, showed only a slight decrease in molecular weights during the reactions.This aligns with previous PET degradation results (Appl Microbiol Biotechnol 2009, 84:227 237) and suggested a predominantly surface-level fragmentation of PET.This is different from the degradation pattern of polyvinyl alcohol, which undergo rapid molecular size reduction because the polymer molecules are solved, dispersed, and uniformly susceptible to enzyme attack.For PET, the hydrolysis process involves both endo-type and exo-type hydrolyses concurrently.Kawai et al. suggested that once the surface ester bonds are depolymerized, monomers such as MHET and BHET are produced through exotype hydrolysis at the ends of fragmented molecules.The sufficiently depolymerized molecules are either released from the PET structure or removed during washing steps preceding GPC analysis, resulting in a marginal change in the overall molecular size of the remaining PET block.Further details of this discussion have been added to the revised manuscript on pages 12-13, lines 417-426, Considering the consistency of the GPC results across all degradation reactions at 65°C for TurboPETase and LCC ICCG , and 72°C for LCC ICCG , and the constraints of the revision timeline, we have only conducted the analyses for Figure 4A with three replicates.We hope this revision aligns with your expectations and addresses your concerns adequately.
: The new Figure 2, comparing the degradation performance of various PETases, is a valuable addition.However, I am surprised that the authors have not cited or discussed a recent publication by Carbios (https://doi.org/10.1021/acscatal.3c02922),which presents similar experiments under industrial conditions but partly with discrepant outcomes regarding the performance ranking of certain enzymes.Including this reference and discussing the discrepancies between different studies would significantly strengthen the manuscript, particularly in the context of positioning TurboPETase as a promising candidate for industrial applications.By addressing these points, the authors will significantly enhance the manuscript's contribution to the field of PET degradation, providing a more robust and comprehensive understanding of this important area of research.
Response: Thanks for highlighting the publication by Carbios, which provides valuable perspectives on enzyme degradation performance under industrially relevant conditions, aligning with the focus of our research.Upon reviewing the article, we observed notable variations in enzyme performance at different substrate concentrations.Specifically, it showed that HotPETase outperformed PES-H1 L92F/Q94Y in specific activity at a lower substrate concentration of 2g L -1 , while at higher concentrations, PES-H1 L92F/Q94Y demonstrated superior degradation conversion.The authors attributed this performance drop in HotPETase to product inhibition and limited thermostability.
As our experiments in Figure 2, conducted at high solids concentrations (30 g kg -1 ), yet still below the industrial standard of 200 g kg -1 , we acknowledge and respect the data reported in the study by Carbios, as it more closely reflects industrial reaction conditions.However, it is worth noting that even under these industrial conditions, PES-H1 L92F/Q94Y 's performance did not surpass that of LCC ICCG .Therefore, we maintain our main conclusion that TurboPETase exhibits superior performance.We have integrated a discussion on this topic into our manuscript, please refer to page 6, lines 206understanding of the performance of various enzymes under different conditions.
Once again, we wish to extend our deepest appreciation for your invaluable and insightful feedback throughout this review process.Your expertise and thorough guidance have played a pivotal role in refining and enriching our study.Each of your suggestions has been carefully considered and integrated, significantly enhancing the depth, rigor, and alignment of our research with the highest standards of the field.This process has undoubtedly elevated the quality and credibility of our work.

Figure R1 .
Figure R1.Comparison of the PET-hydrolytic activity of the M6 variants towards Gf-PET films in different buffer concentrations.Reactions were performed at 65 °C using 30 g kg -1 solids loading and 2 mgemzyme gPET -1 enzyme loading for 3 hours.

Fig. 5
Fig. 5 to calculate normalized depolymerization curves based on combinatorial data obtained by NaOH consumption, HPLC analysis of the formation of TPA/MHET/BHET; EG, and the weight loss determinations, with the original data presented separately in a table.
R1-R2.Regarding the Figure S14 of PBT degradation and Figure S15 of dual enzyme degradation, we have introduced a dedicated section in the revised manuscript, titled -enzym to page 11, lines 347-Previous study has demonstrated limited biodegradation efficiency of

Table R2 .
Crystallinity and molecular mass of PET materials before and after pretreatments determined by DSC and GPC, respectively.Mw: weight average molecular mass, Mn: number average molecular mass.
Counterparts like BhrPETase, LCC, LCC ICCG , ICCG I6M , and PES-H1 L92F/Q94Y which exhibit high degradation performance at elevated temperatures, rendered hydrolytic activity 1.8-, 4.7-, 2.1-, 2.0-, and 19fold lower than that of TurboPETase, respectively.At suboptimal temperatures, TurboPETase consistently outperformed other PET hydrolases, albeit the decreased hydrolytic activity.At the optimal temperature of HotPETase and CaPETase M9 of 60 °C, TurboPETase generated 11.73 mM monomer product in 3 h, whereas 8: Statements in lines 20-21 and 356-357 appear to be exaggerated.At least in refs.30and32, as well as the omitted publication by Ding et al. (see above for DOI), additional enzymes or mutants that outperform LCC-ICCG under specific reaction conditions, have been demonstrated.Obviously, these enzymes are not included in this study for experimental comparison with TurboPETase, nor are they even given serious consideration.As a highly active research field, additional promising enzyme variants may be published during this manuscript's revision stage.Before overselling their own findings, I strongly advise the authors to remain open to learning about the most recent developments in this research field.Response: Thanks for the valuable comment that leads us to perform additional experiments to achieve more comparative analysis with other benchmark PETases.The realm of PETase under elevated buffer concentration.Across these conditions, TurboPETase consistently manifested an enhanced PET-hydrolytic activity compared to the other PET hydrolases.Details are shown in revised Figure2and Supplementary Fig.S6in the revised manuscript, and please also refer to page 6, lines 181-205,